The influence that some non-natural amino acids have on the structural and biological activity of peptides has been briefly studied. For example, Moore et al. (Can. J. Biochem. 1978, 56, 315) disclosed the effect of the basic amino acid side chain length and the penultimate residue on the hydrolysis of benzoyldipeptides by carboxylicpeptidase B1 (CPB). Non-natural amino acids including homolysine and homoarginine were incorporated into small peptide chains, and the kinetic parameters were determined for the CPB catalyzed hydrolysis of the peptide. Also, Lindeberg et al. (Int. J. Peptide Protein Res. 1977, 10, 240) disclosed the synthesis of 1-deamino-4-L-valine-8-DL-homolysine-vasopressin and protected 1-deamino-4-L-valine-8-D-lysine-vassopressin in which non-natural amino acids were incorporated. The non-natural amino acids were formed by addition of a methylene group to lysine and arginine to generate the non-natural amino acids homolysine and homoarginine, respectively. The study revealed that peptides with homolysine and homoarginine reduced the antidiuretic activity of the peptides.
Naturally occurring endogenous peptides are ideal drug candidate leads by virtue of their myriad activities in promoting and regulating biological processes. Inherent in the chemistry and biology of peptides, however, are several factors that also make them poor drug candidates. Peptides most often exert localized effects and are rapidly degraded within the body. In addition, most peptides are unable to cross biological membranes, including the small intestine and blood brain barrier (BBB). Finally, peptides often bind to more than one receptor or receptor subtype, thus rarely showing the selectivity required of a viable drug candidate. Therefore, for a peptide to become a viable drug candidate, improvements in blood stability, receptor selectivity, and barrier crossing should be made without eliminating inherent binding affinity.
Numerous strategies have been developed as methods for improving peptide stability, including N- and C-terminal modifications to prevent exopeptidase activity, amide backbone modifications, and the introduction of conformational constraints to disguise peptides from peptidase degradation. Other therapeutic compounds employ a prodrug moiety intended to modify its overall hydrophobicity, which can result in the compound crossing biological membranes. In this case, the compound is cleaved into its active component by endogenous enzymes. While each of these strategies has been used to improve peptides as drug candidates, a universal solution for creating stable, receptor-selective peptides that cross biological barriers has not been discovered.
Consequently, there is a need in the art for non-natural amino acids and for peptides incorporating such acids to achieve superior effects, such as, for example, improved diagnostic or disease fighting activity. Thus, the non-natural amino acid concept could be applied to development of new peptide pharmaceuticals. One example of such a development is the application to neuropeptides such as neurotensin.
SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Variables, such as R1-R3, n, z, X, Y, Cα and Cβ, throughout the application are the same variables as defined herein unless stated to the contrary.
The term “alkyl” as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred alkyl groups herein contain from 1 to 6 carbon atoms.
The term “alkenyl” as used herein refers to a hydrocarbon group of 2 to 24 carbon atoms, with preferred groups within this class containing 2 to 6 carbon atoms, and structural formula containing a carbon-carbon double bond.
The term “alkynyl” as used herein refers to a hydrocarbon group of 2 to 24 carbon atoms, with preferred groups within this class containing 2 to 6 carbon atoms, and a structural formula containing a carbon-carbon triple bond.
As used herein, especially in reference to alkyl, alkenyl and alkynyl, unless defined otherwise, the term “lower” refers to a moiety having from 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms.
The term “alkylating agent” as provided herein is a compound with the structural formula RX, where R is an alkyl, alkenyl or alkynyl group as previously described, and X, which is preferably a halide such as chloride, bromide or iodide.
As used herein, the term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., selectivity, stability) when the non-natural amino acid is either substituted for a natural amino acid unit of a peptide or otherwise incorporated into a peptide.
As used herein, the term “peptide” refers to a class of compounds composed of amino acids chemically bound together. In general, the amino acids are chemically bound together via amide linkages (—CONH—); however, the amino acids may be bound together by other chemical bonds known in the art. For example, the amino acids may be bound by amine linkages. Peptide as used herein includes oligomers of amino acids and small and large peptides, including polypeptides.
As used herein, the term “activity” refers to a biological activity.
As used herein, the term “pharmacological activity” refers to the inherent physical properties of a peptide or polypeptide. These properties include but are not limited to half-life, solubility, and stability and other pharmacokinetic properties.
The term “organic acid salt” as used herein refers to the salt form of an amine group with an alkyl or aryl C1-C9 carboxylic, sulfonic, or phosphoric acid.
The term “inorganic acid salt” as used herein refers to the salt form of an amine group with a mineral acid such as hydrochloric, sulfuric, sulfonic, phosphoric, nitric, nitrous, or hydrobromic acid.
The term “aromatic of C6 to C18” as used herein refers to an aromatic hydrocarbon such as phenyl, naphthyl, anthracenyl, or an arylalkyl hydrocarbon such as benzyl, phenethyl or naphthylmethylenyl.
The term “heteraromatic of C4 to C18 and of one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination” as used herein refers to a heteroaromatic hydrocarbon containing one or two heteroatoms or an alkyl heteroaromatic hydrocarbon such as thienyl, furyl, pyrrolyl, azathienyl, azafuryl, pyridinyl, thiapyridinyl, pyrazinyl, methylenylpyridinyl, ethylenylpyridinyl, methylenylpyrrolyl and the like.
The chemical, pharmaceutical and biological terms used herein follow the ordinary and customary meanings one of skill, such as a Ph.D. researcher in the field would attribute to them. Such meanings may be found in appropriate technical dictionaries and treatises such as but not limited to “Hawley's Condensed Chemical Dictionary”, 11th Ed., Sax and Lewis Editors, Van Nostrand Reinhold Publishing, New York, N.Y. 1987; “Concise Chemical and Technical Dictionary”, 4th enlarged Ed. Bennett Editor, Chemical Publishing Inc., New York, N.Y., 1986, “The Merck Index” 11th and succeeding Editions, Merck & Co. Rahway, N.J. 1989 and more recent; “Advanced Organic Chemistry” 4th Ed., J. March, Wiley Interscience, New York, N.Y. 1992; “Concise Dictionary of Biomedicine and Molecular Biology”, Pei-Show Juo Ed., CRC Press, New York, N.Y. 1996; “Molecular Cell Biology”, Daniell, Lodish, Baltimore, Scientific American Books, New York, N.Y. 1986; the disclosures of all of these dictionaries and treatises are incorporated herein by reference.
The present invention concerns alpha-desamino, alpha-alkyl amino acid compounds (desamino, alkyl amino acid compounds) that are capable of carrying positively charged side chains, their synthesis, their application as substitutes for natural amino acid moieties of biologically active peptides and the resulting peptides as well. In particular, alpha-alkyl, alpha-desamino arginine, lysine and ornithine as well as their substituted and derivatized side chain analogs constitute preferred embodiments of the invention. These desamino, alkyl amino acid compounds can be substituted for arginine and/or lysine moieties in any known, biologically active peptide such that the substituted peptide will be truncated at the substitution position. Alternatively, these desamino, alkyl amino acid compounds can be coupled to the amino group of the N-terminus of any known biologically active peptide to produce an extended peptide. The truncated and extended peptides have significant biological selectivity and biological half lives owing to their resistance toward amino peptidase degradation.
In a first aspect, the invention relates to a non-natural desamino, alkyl amino acid compound having Formula I:

wherein
n is an integer of from 0 to 5, preferably, 2 to 5;
m is zero or an integer of 1;
R is a straight or branched chain alkyl group of C1-C6, or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
R1, R2, and R3 are, independently, hydrogen or branched or straight chain alkyl, alkenyl or alkynyl of C1-C6 or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination and with the proviso that a maximum of two of R1, R2, and R3 may be selected to be the aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic group;
Cα is a carbon atom having either R or S stereochemistry;
or an ester, amide, alkyl amide or metal cation or ammonium salt of the carboxylic acid group thereof, or an organic or inorganic acid salt of the amine group thereof, or any combination thereof.
In a second aspect, the invention relates to a non-natural desamino, alkyl amino acid compound of the formula II:

wherein
n is an integer of from 0 to 6, preferably, 2 to 5;
when dashed line a is not present, X and Y are independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C6;
when dashed line a is present, X—Y is (CH2)z, wherein z is an integer of from 1-8, preferably, 2 to 4;
R is a straight or branched chain alkyl group of C1-C6, or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
R4 is hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C6, or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, and;
Cα is a carbon atom and the stereochemistry at Cα is either R or S;
or an ester, amide, alkyl amide or metal cation or ammonium salt of the carboxylic acid group thereof, or an organic or inorganic acid salt of the amine group thereof, or any combination thereof.
A third aspect of the present invention relates to a non-natural desamino, alkyl amino acid compound of the formula III:

wherein
n is an integer of from 0 to 5, preferably, 2 to 5;
X—Y is (CH2)z, wherein z is an integer of from 0 to 6, preferably, 2 to 4;
R is a straight or branched chain alkyl group of C1-C6, or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
R6, and R7 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C6, or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination; and
Cα is a carbon atom and the stereochemistry at Cα is either R or S;
or an ester, amide, alkyl amide or metal cation or ammonium salt of the carboxylic acid group thereof, or an organic or inorganic acid salt of the amine group thereof, or any combination thereof.
A fourth aspect of the invention relates to a non-natural desamino, alkyl amino acid compound of the formula IV:

wherein
n is an integer of from 0 to 5, preferably, 2 to 4;
R is a straight or branched chain alkyl group of C1-C6, or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination;
R9, R10, and R11 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C6, or an aromatic group of C6-C18 or a corresponding substituted aromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination, or a heteroaromatic group of C4-C18 and one or two heteroatoms selected from oxygen, sulfur and nitrogen in any combination or a corresponding substituted heteraromatic group with one or two substituents selected from halogen, alkyloxy, carboxy, amide or alkyl in any combination and with the proviso that a maximum of two of R9, R10, and R11 may be selected to be the aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic group; and
Cα is a carbon atom and the stereochemistry at Cα is either R or S;
or an ester, amide, alkyl amide or metal cation or ammonium salt of the carboxylic acid group thereof, or an organic or inorganic acid salt of the amine group thereof, or any combination thereof.
A further aspect of the invention relates to the addition of the non-natural desamino, alkyl amino acid compounds of the invention to the N-terminus amino group of biologically active peptides or their substitution for naturally occurring congener amino acid moieties of biologically active peptides. Preferred congener moieties include arginine and/or lysine.
The addition to the N-terminus amino group of a known, biologically active peptide provides an extended peptide that has selective, long lasting biological activity of the same kind as the known, biologically active peptide. The addition can be accomplished by known methods for coupling acid and amine groups together to form amide bonds, including use of acyl azide coupling, carbodiimide coupling, acid ion exchange resin, triaminoboranes and enzyme coupling. A preferred method involves the use of an amino exopeptidase under conditions that promote the peptide bond formation. In some embodiments of the invention, the semisynthetic peptides are produced by substituting a non-natural amino acid compound for the N-terminal arginine residue of NT (8-13), e.g., ABS201.
Preferred embodiments of the peptides on which the extended peptides are based include biologically active peptides useful for treatment or prevention of malconditions. A list of preferred categories and examples is included in the sections below. Some preferred categories include but are not limited to transcription factors, ligands for cellular receptors, hormones and extracellular binding peptides. Some preferred examples include but are not limited to enkephlin, LHRH and analogs, neuropeptides, glycoincretins, integrin and analogs, glucagons and glucagon-like peptides, antithrombotic peptides, cytokines and interleukins, transferrins, interferons, endothelins, natriuretic hormones, extracellular kinase ligands, angiotensin enzyme inhibitors, peptide antiviral compounds, thrombin, substance P, substance G, somatotropin, somatostatin, GnRH and analogues, secretin, bradykinin, vasopressin and analogues, insulin and analogs thereof, growth factors, as well as others. The extended peptide is formed by coupling the N-terminus amino group of a basis peptide to the carboxyl group of a desamino, alkyl amino acid compound of the invention.
The substitution of desamino, alkyl amino acid moiety for an arginine or lysine moiety of a biologically active peptide provides a truncated peptide having selective, long-lasting biological activity. Any known biologically active peptide having an arginine and/or lysine moiety within its amino acid sequence can serve as the basis for the corresponding truncated peptide. Beginning at that ARG or LYS moiety, the truncated peptide will have the same downstream sequence as the known, biologically active peptide but the upstream sequence will be absent. In addition, that ARG or LYS moiety will be exchanged for a desamino, alkyl amino acid moiety, thus providing the truncated peptide. Several known biologically active peptides are penultimately formed as pro-peptides with an arginine or lysine moiety at the pro-peptide or precursor cleavage position, or are formed as final peptides containing an arginine or lysine moiety at a position that can be cleaved to provide an active truncated peptide. Trypsin is an enzyme specific for such cleavage points. Examples include glucagon-like peptide, neurotensin, proinsulin, and thrombin. The truncated versions of these examples with a desamino, alkyl amino acid compound substituted for the arginine or lysine moiety provide selective, long-lasting biological activity.
A further aspect of the invention includes pharmaceutical and cosmetic compositions of the desamino, alkyl amino acid compound, of the extended or truncated peptide, and combinations thereof. Unit dosage forms and biologically effective formulations of the pharmaceutical compositions are included. The cosmetic formulations include appropriate oil, creme, wax or aqueous base cosmetic carriers.
Yet another aspect of the invention includes methods of screening, diagnosis and treatment using the desamino, alkyl amino acid compounds of the invention and/or the addition or truncated peptides.
One embodiment of the invention is a truncated neurotensin peptide having a desamino, alkyl amino acid as its N-terminus amino acid moiety.
The invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of the inventions, such as compounds of Formula I, II, III, and/or IV and peptides that contain such compounds. One class of such intermediates includes the N-protected or carboxyl protected or N— and carboxyl protected compounds of Formulas I, II, III and IV. These protected intermediates are described in detail in the following sections of the application. Another class of such intermediates includes the carboxylate salts of the compounds of Formulas I, II, III and IV, the organic or inorganic acid amine salts of those compounds and the double salts (carboxylate, amine salts).